This invention relates to an apparatus for fusing cells by applying an electrical stimulus to them.
Generally, the apparatus comprises a chamber enclosing a pair of spaced electrodes between which a suspension containing cells of two different species to be fused together are put, a source of electrical stimulus, means for applying an electrical stimulus to the cells through the electrodes, and a controller for controlling the application of the electrical stimulus to the cells.
As the electrical stimulus an alternating current (AC) field and a direct current (DC) pulse are used, and as the electrodes a pair of wire or plate electrodes are used.
For effecting cell fusion an AC field is first applied through the electrodes to the cells suspended in a solution put between the electrodes to cause close membrane contact between adjacent cells, and then a DC pulse of a relatively high electric field is applied to the cells to fuse them together.
If a pair of wire electrodes are used, the following problems are encountered:
(1) The strength of the electric field between the electrodes is not uniform, so that it is difficult to accurately know the effective strength of the electric field applied to the cells.
(2) The fused cells adhere to the electrodes, so that when they are separated from the electrodes, they are damaged or broken.
(3) Multi-fusion occurs, that is, three or more cells are linked like a pearl chain.
There is also known an apparatus which uses a pair of parallel plate electrodes to which a DC voltage is applied for fusing the cells that fill the space between the electrodes. In this apparatus, beside the above problems (2) and (3) there is a third problem that the degree of membrane contact between adjacent cells is low and consequently the rate of fusion is low.
Accordingly, it is one object of the invention to provide an apparatus for fusing cells which can provide a substantially uniform field strength which is an important factor for cell fusion, so that it is possible to exactly know the effective strength of the electric field applied to the cells to be fused together, and wherein the cells do not stick to the electrodes but closely contact each other between the electrodes, so that it is possible to effect fusion of a single pair of cells without causing multi-fusion wherein many cells are fused together.
The cells to be fused are suspended in a solution, and the suspension is put in a space between a pair of opposed electrodes in a fusion chamber. If the amount of the suspension is relatively small such as, for example, 0.05 ml. small electrodes suffice and the chamber enclosing the electrodes can be of a simple shape and construction so that it is relatively easy to handle the chamber.
If the amount of the cell suspension, however, is relatively great such as, for example, 1 ml, the space between the electrodes and/or the length of the electrodes must be increased to accommodate the suspension. If the space between the electrodes is increased, the voltage applied to the electrodes must be increased, with resulting increase in the size of the instrument as a whole. If the length of the electrodes is increased, in order for a fusion chamber of a limited size to accommodate the elongated electrodes, the electrodes must be of a complicated shape. If long linear electrodes are used, the chamber must be elongated, with resulting decrease in the easiness of manipulation of the instrument.
Accordingly, it is another object of the invention to provide a fusion chamber which is small in size and simple in construction and yet capable of containing a sufficient amount of a suspension containing cells to be fused together.
Generally, when cells are fused by application of an electrical stimulus to them, it takes about 30 minutes for the fusion to be completed. During the process the ambient temperature and application of the electric field cause the temperature of the cell suspension to rise so that evaporation occurs to cause the osmotic pressure of the cell suspension to increase with resulting destruction of the cells.
Accordingly, it is still another object of the invention to provide a fusion chamber which can prevent excessive evaporation and decrease of the cell suspension.